HPLC in Peptide Purity Analysis: What the Published Literature Reports

High-performance liquid chromatography (HPLC) appears throughout the analytical literature as the default separation technique for assessing synthetic peptide purity, and published method papers describe several distinct purity questions it is used to address: chemical purity of a crude or purified peptide, chiral (enantiomeric) purity of constituent amino acids, impurity and new-peak detection in biopharmaceutical release testing, and radiochemical purity of radiolabelled peptide probes. Chiral purity is one of the longest-studied applications. An analytical method study described hydrolysing peptides in deuterated hydrochloric acid/acetic acid, derivatising the liberated amino acids with Marfey's reagent, and resolving D-/L-diastereomeric pairs in a single reversed-phase HPLC-electrospray ionisation-mass spectrometry separation; the deuterated hydrolysis step was reported as necessary because acid hydrolysis itself racemises amino acids, and deuterium incorporation at the alpha-carbon marks which residues racemised during sample preparation [1]. That approach was applied in vitro to determine the chiral purity of each amino acid in a hexapeptide by-product isolated from a kilogram batch of synthetic thymopentin [1]. A later in vitro method development paper reported a direct chiral HPLC-ESI-MS/MS method that also used deuterated-acid hydrolysis but omitted derivatisation and multiple external standards, with reported method validation feasibility across nineteen chiral proteogenic amino acids and recovery of D-amino acid substitutions at each residue of an octapeptide over a 0.1–1.0 % range; results on four model peptides of 8–14 residues were reported as comparable to traditional testing methods [2]. Several synthesis papers use HPLC as the reported purity readout for chemically prepared peptides. An in vitro chemistry study on a fibre polyacrylamide resin (amino-Li-resin) for solid-phase peptide synthesis reported crude purity of several model peptides, including difficult sequences and hindered amino acids, as assessed by HPLC analysis [3]. A total chemical synthesis study of disulfide-rich starfish relaxin-like peptides reported that the reduced, unfolded A-chain of one peptide produced disordered RP-HPLC profiles that initially resembled synthesis failure, and that oxidation markedly improved the chromatographic profile, leading the authors to attribute the poor trace to peptide aggregation rather than failed synthesis; both target peptides were ultimately reported at purities exceeding 95 % [4]. This is an in vitro analytical observation about how sample state can confound a purity chromatogram [4]. In a peptide conjugation study, reversed-phase HPLC was used to characterise PEGylated leuprolide conjugates and was reported to indicate high purity of the resulting conjugates, alongside MALDI-MS evidence of 1:1 PEGylation, with subsequent in vitro cell-line bioactivity work and in vivo pharmacokinetic measurements in rats [5]. A biomaterials study of a collagen-targeting bi-functional peptide reported sequence confirmation by nano-LC-MS/MS, greater than 90 % purity by HPLC, and a single 4434.5 Da band by SDS-PAGE and MALDI-TOF as part of its in vitro structural characterisation, prior to rat model work [6]. A food-allergen study likewise reported that the purity of three synthesised overlapping peptides was identified by mass spectrometry and HPLC at more than 90 % before immunological testing [7]. An older characterisation review reported that synthetic canine CCK-58, purified by reversed-phase HPLC, was characterised by isocratic and gradient HPLC alongside amino acid analysis, mass spectral analysis and sequence analysis, and matched natural canine CCK-58 in elution profile, composition, sequence and mass [8]. For protein biopharmaceuticals, published work describes HPLC-based purity assays being displaced or supplemented by LC-MS peptide mapping. A method development and qualification study reported that the multi-attribute method by LC-MS peptide mapping has the potential to replace multiple conventional HPLC- and capillary electrophoresis-based purity/impurity assays for release and stability testing, and described a new peak detection workflow validated according to ICH Q2 guidelines that detected relevant peptide species below 1 % relative abundance without reporting false positive peaks in drug product stability and drug substance impurity case studies [9]. A separate review of HPLC-MS in proteomics described reversed-phase, hydrophilic interaction, hydrophobic interaction, ion-exchange and size-exclusion chromatography as the commonly used separation modes, and noted that no single chromatographic or electrophoretic technology has the peak capacity to resolve highly complex mixtures into individual components, motivating multidimensional LC-MS strategies [10]. The literature also documents disagreement between HPLC purity values and orthogonal techniques. A capillary zone electrophoresis (CZE) method study reported that a commercial peptide nucleic acid preparation was evaluated at about 81.4 % purity by CZE, lower than the supplier's RP-HPLC-derived value of 99.9 % and also lower than the 94.8 % the same group determined by RP-HPLC, with the CZE run taking about 5 minutes and 90 nL of sample versus about 20 minutes and 20 µL for RP-HPLC [11]. Reviews of capillary electrophoresis in pharmaceutical analysis have described CE as complementary to HPLC for purity and structural confirmation of peptides and proteins and for chiral separations [12], and a later review argued that CE is a reasonable alternative to HPLC for impurity profiling and enantiomeric purity but is rarely applied in drug quality control [13]. A forensic case report illustrates a different limitation: powder from vials labelled as the peptide melanotan II produced no library match by HPLC-DAD, and identification required mass spectrometric analysis against a reference standard, after which the purity of the seized material was determined to be 30 % [14]. In radiopharmaceutical chemistry, HPLC is reported as the radiochemical purity readout. A PET probe study reported that 68Ga-DOTA-TFpep was confirmed by radio-HPLC at greater than 98 % purity with high stability in PBS, followed by in vitro cell uptake work and in vivo biodistribution and imaging in mouse xenograft models [15]. A radiolabelling study of an anti-HER2 single-chain variable fragment reported the use of TLC and HPLC among its characterisation methods, with radiochemical purity of around 98 % and reported in vitro stability for at least 24 hours in PBS, saline, human plasma proteins and histidine solution [16]. An 18F-labelling study of a 13-amino-acid transglutaminase-2-targeting peptide reported HPLC purification of both the radiolabelling synthon and the putative radiotracer, and noted that earlier 11C- and 18F-labelled probes for this target had not found widespread application partly because of purity and metabolism issues; imaging was performed in mice bearing xenografts [17]. A comparative quality-control study in hospital radiopharmacy compared radio-HPLC with single-strip and two-strip thin-layer chromatography methods for [99mTc]Tc-MAG3 and reported statistically significant differences in measured radiochemical purity between one TLC method and radio-HPLC, as well as significant differences in analysis duration between the four procedures [18]. Chromatographic purity also features in preparative and food-science contexts. A review of egg protein fractionation described liquid chromatography as the most commonly used technique for obtaining individual proteins at high recovery and purity [19], and a review of bioactive compounds from natural sources described HPLC and GC-MS among advanced purification methods enabling recovery of high-purity bioactives including peptides [20].